The present invention relates to a method for producing shikimic acid. Shikimic acid is useful for intermediates of aromatic compounds synthesis such as phenylalanine, tyrosine, tryptophane, p-aminobenzoic acid or p-hydroxybenzoic acid. Shikimic acid is also used as component of waste disposal material and the like.
Shikimic acid that is an aromatic intermediate is synthesized in four enzymatic reactions from phosphoenol pyruvate and erythrose-4-phosphate. These four enzymes are encoded by aroA, aroB, aroC, and aroD genes in Bacillus subtilis. Shikimic is converted into chorismic acid by enzymatic reactions by aroI, aroE and aroF. The pathway from phosphoenol pyruvate and erythrose-4-phosphate to chorismic acid is called as shikimic acid pathway. Shikimic acid pathway is also known as a common pathway for biosynthesis of aromatic amino acids L-tryptophane, L-phenylalanine and L-tyrosine.
Shikimic acid is obtained from plants heretofore, and it has not been produced by direct fermentation using microorganism.
Bacillus subtilis 1-118 (aroI116, amy4), which has been known as amylase-deficient strain, is known to have a mutation in aroI gene (Yuki, S., Japan. J. Genetics, 50(2), 155-157 (1975)). However, it is not known that the strain 1-118 produces shikimic acid.
Although, it also has been known that Bacillus subtilis SB130 (aroE130, hisH32) has a mutation in a gene coding for 5-enolpyruvylshikimate-3-phosphate synthase (EC:2.5.1.19), it is not known that the strain produces shikimic acid.
An object of the present invention is to provide a method for producing shikimic acid by direct fermentation and a microorganism that is used in the method.
As a result of diligent and repeated investigation in order to achieve the object described above, the present inventors determined that strains of B. subtilis, carrying defective shikimate kinase enzyme accumulated shikimate. Further, the present inventor succeeded in improving shikimic acid productivity of the bacterium by enhancing an activity of shikimate dehydrogenase and an activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (sometimes referred to as xe2x80x9cDAHP synthasexe2x80x9d).
Further, the present inventors found that the shikimic acid productivity of B. subtilis strain can be increased by depletion of aroE gene product, 5-enolpyruvylshikimate-3-phosphate, and enhancing the activity of shikimate dehydrogenase synthase, even if the strain possesses active shikimate kinase.
That is, aspects of the present invention are as follows:
(1) A method for producing shikimic acid, comprising the steps of cultivating a bacterium belonging to the genus Bacillus which is deficient in shikimate kinase activity and has shikimic acid productivity in a medium, producing and accumulating shikimic acid in the medium, and collecting shikimic acid from the medium;
(2) The method of (1), wherein the bacterium is Bacillus subtilis; 
(3) The method of (1), wherein shikimate dehydrogenase activity in the cell of the bacterium is enhanced;
(4) The method of (1), wherein shikimate dehydrogenase activity is enhanced by increasing copy number of a gene encoding shikimate dehydrogenase, enhancing expression regulation sequence of the gene or integrating the gene into chromosomal DNA of the bacterium;
(5) The method of (4), wherein the shikimate dehydrogenase gene, in which its inherent promoter is displaced by a promoter of other gene or to which a promoter of other gene is added, is integrated into the chromosomal DNA;
(6) The method of (5), wherein activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase in the cell of the bacterium is further enhanced;
(7) The method of (6), wherein activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase is enhanced by increasing copy number of a gene encoding 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, enhancing expression regulation sequence of the gene or integrating the gene into chromosomal DNA of the bacterium;
(8) A bacterium belonging to the genus Bacillus having shikimic acid productivity, wherein the bacterium is deficient in shikimate kinase activity, and shikimate dehydrogenase activity in the cell of the bacterium is enhanced; and
(9) The bacterium of (8), wherein activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphatesynthase in its cell is enhanced.
(10) A method for producing shikimic acid, comprising the steps of cultivating a bacterium belonging to the genus Bacillus which is deficient in 5-enolpyruvylshikimate-3-phosphate synthase activity and has shikimic acid productivity in a medium, producing and accumulating shikimic acid in the medium, and collecting shikimic acid from the medium.
(11) The method of (10), wherein said bacterium is Bacillus subtilis. 
(12) The method of (10), wherein shikimate dehydrogenase activity in the cell of said bacterium is enhanced.
(13) The method of (12), wherein shikimate dehydrogenase activity is enhanced by increasing copy number of a gene encoding shikimate dehydrogenase, enhancing expression regulation sequence of said gene or integrating said gene into chromosomal DNA of said bacterium.
(14) A bacterium belonging to the genus Bacillus having shikimic acid productivity, wherein said bacterium is deficient in 5-enolpyruvylshikimate-3-phosphate synthase activity, and shikimate dehydrogenase activity in the cell of said bacterium is enhanced.
In the present invention, the term xe2x80x9chaving shikimic acid productivityxe2x80x9d refers to an activity to accumulate shikimic acid in a medium, when the bacterium of the present invention is cultivated in the medium.
The present invention will be explained in detail below.
A bacterium belonging to the genus Bacillus of the first embodiment of the present invention is deficient in shikimate kinase activity. As the bacterium belonging to the genus Bacillus, there are exemplified Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus.
A bacterium belonging to the genus Bacillus deficient in shikimate kinase activity is exemplified by a mutant which substantially completely loses shikimate kinase activity and a mutant of which shikimate kinase activity is significantly lower than that of wild type strain. Alternatively, a bacterium belonging to the genus Bacillus may be a strain in which a shikimate kinase gene (aroI) on its chromosome is disrupted by homologous recombination.
Depletion of shikimate kinase activity of the bacterium belonging to the genus Bacillus may be achieved by the conventionally known mutation treatment and selection of mutant strain which has a mutation in its gene coding for the enzyme (aroI). The mutation treatment includes a method for treating the bacterium belonging to the genus Bacillus with ultraviolet irradiation or a mutating agent such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment.
An aroI mutant of the bacterium belonging to the genus Bacillus may be also obtained by gene conversion method using aroI mutant of a bacterium belonging to the genus Bacillus. That is, a wild type aroI gene on a plasmid is converted to a mutant aroI gene by gene conversion method using the plasmid carrying a cloned aroI gene and a mutant strain having a mutation in its aroI gene. Then a wild type aroI gene in a bacterium belonging to the genus Bacillus is converted to the mutant aroI gene.
Concretely, a bacterium belonging to the genus Bacillus is exemplified by Bacillus subtilis 1-118 (aroI116, amy4) and I-116 which was derived from the strain 1-118 (see after-mentioned Example 1) or the like. The strain 1-118 is described in Yuki, S., Japan J. Genetics, 50 (2), 155-157 (1975).
A bacterium belonging to the genus Bacillus of the present invention may be preferably enhanced in shikimate dehydrogenase activity. Furthermore, it is preferable that the bacterium is further enhanced in DAHP synthase. Shikimate dehydrogenase activity or DAHP synthase activity in a bacterial cell can be enhanced by, for example, increasing the copy number of genes coding for these enzymes or integrating the genes into chromosomal DNA of the bacterium.
Concretely, enhancement of shikimate dehydrogenase activity may be achieved by constructing recombinant DNA by ligating a gene fragment that codes for shikimate dehydrogenase with a vector that functions in bacteria belonging to the genus Bacillus, transforming a host strain of bacterium belonging to the genus Bacillus by introducing the recombinant DNA to the strain. The shikimate dehydrogenase is coded by aroD gene.
As to the aroD gene, the gene of bacterium belonging to the genus Bacillus and also the gene derived from other organisms can be used.
As for plasmids which are used for introducing aroD gene into cells of bacteria belonging to the genus Bacillus, any plasmid that is replicable in cells of the bacteria, which is concretely exemplified by pUB110, pC194, pE194, pSM19035, pMX30, pMX39, pCB20, pCB30 and pCA1 and the like. Alternatively, the bacteria belonging to the genus Bacillus may be transformed by integrating a linearized DNA into chromosomal DNA instead of using a vector.
In order to prepare recombinant DNA by ligating the aroD gene and a vector that can function in a cell of bacterium belonging to the genus Bacillus, the vector is digested by restriction enzyme(s) corresponding to the termini of a DNA fragment comprising the aroD gene. Ligation is generally performed by using a ligase such as T4 DNA ligase.
To introduce the recombinant DNA prepared as described above to bacterium belonging to the genus Bacillus, any known transformation methods can be employed. For instance, employable are a method of preparing competent cells from cells which are at the growth phase followed by introducing the DNA thereunto [see Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)]. In addition to this, also employable is a method of making DNA-recipient cells into the protoplast which can easily take up recombinant DNAs followed by introducing the recombinant DNA into the cells [see Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979)]. Integrating the aroD gene into chromosomal DNA of a bacterium belonging to the genus Bacillus may be achieved by a congression method employing linearized DNA in which two sequences are randomly integrated into chromosomal DNA (Erickson, R. T. et al., Genetics, 73(1), 13 (1973); Nester, E. W. et al., Genetics, 48, 529 (1963)).
Other than the above-described gene amplification, enhancement of shikimate dehydrogenase activity can also be achieved by substituting the expression regulation sequence such as promoter of the aroD gene with a more potent one. The expression of aroD gene can be enhanced by introducing a recombinant gene in which aroD structural gene is ligated with a strong promoter into cells of bacteria belonging to the genus Bacillus. Substitution of the expression regulation sequence of aroD gene on the chromosomal DNA may be achieved by the method described in Japanese Patent Laid-Open Publication No. 1-215280. As potent promoters, for example, Pr(rpmA) which is the promoter of the ribosomal protein gene (rpmA) of Bacillus amyloliquefaciens is known. By substituting the promoter inherent in aroD gene with these promoters, the expression of aroD gene is enhanced, thereby enhancing shikimate dehydrogenase activity. The enhancement of the expression regulation sequence may be combined by increasing the copy number of aroD gene.
DAHP synthase activity can be enhanced according to the above-described methods in which a gene encoding DAHP synthase (aroA gene) is used in place of aroD gene.
A bacterium belonging to the genus Bacillus of the second embodiment of the present invention is deficient in 5-enolpyruvylshikimate-3-phosphate synthase activity. As the bacterium belonging to the genus Bacillus, there are exemplified Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus.
A bacterium belonging to the genus Bacillus deficient in 5-enolpyruvylshikimate-3-phosphate synthase activity is exemplified by a mutant which substantially completely loses 5-enolpyruvylshikimate-3-phosphate synthase activity and a mutant of which 5-enolpyruvylshikimate-3-phosphate synthase activity is significantly lower than that of wild type strain. Alternatively, a bacterium belonging to the genus Bacillus may be a strain in which a gene coding for 5-enolpyruvylshikimate-3-phosphate synthase (aroE) on its chromosome is disrupted by homologous recombination.
Depletion of 5-enolpyruvylshikimate-3-phosphate synthase activity of the bacterium belonging to the genus Bacillus may be achieved by the conventionally known mutation treatment and selection of mutant strain which has a mutation in its gene coding for the enzyme (aroE). The mutation treatment includes a method for treating the bacterium belonging to the genus Bacillus with ultraviolet irradiation or a mutating agent such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment.
An aroE mutant of the bacterium belonging to the genus Bacillus may be also obtained by gene conversion method using aroE mutant of a bacterium belonging to the genus Bacillus. That is, a wild type aroE gene on a plasmid is converted to a mutant aroE gene by gene conversion method using the plasmid carrying a cloned aroE gene and a mutant strain having a mutation in its aroE gene. Then a wild type aroE gene in a bacterium belonging to the genus Bacillus is converted to the mutant aroE gene.
In the present invention, the bacterium belonging to the genus Bacillus which is deficient in 5-enolpyruvylshikimate-3-phosphate synthase is preferably enhanced in shikimate dehydrogenase activity. Furthermore, it is preferable that the bacterium is further enhanced in DAHP synthase. Shikimate dehydrogenase activity or DAHP synthase activity in the bacterial cell can be enhanced in the same manner as the bacterium used in the first embodiment of the present invention.
In a bacterial cell shikimic acid is converted to shikimate 3-phosphate by action of shikimate kinase enzyme. Shikimic acid was found to be a product inhibitor of shikimate dehydrogenase in B. subtilis, like as in E. coli cells. On the other side, shikimate 3-phosphate do not inhibit shikimate dehydrogenase. The inventors supposed that the feedback inhibition of shikimate dehydrogenase by shikimic acid may be desensitized by increasing shikimate kinase activity, since shikimate kinase reduces an intracellular concentration of shikimate.
Then the inventors tested possibility to construct a shikimate producer carrying a gene encoding an active shikimate kinase. As a result it was found that Bacillus subtilis having the active shikimate kinase gene exhibits an activity to produce shikimic acid by deleting 5-enolpyruvylshikimate-3-phosphate synthase, and the productivity is increased by further enhancing the shikimate dehydrogenase activity. That is, the accumulating shikimate 5-phosphate formed by shikimate kinase are easily dephosphorylated by action of phosphatases and the resulting shikimic acid is exported from bacteria.
Shikimic acid can be produced by cultivating in a medium a bacterium belonging to the genus Bacillus having shikimic acid productivity, which is obtainable as described above, producing and accumulating shikimic acid in the medium, and collecting shikimic acid from the medium.
The medium to be used for cultivation is an ordinary medium containing a carbon source, a nitrogen source, organic ions and optionally other organic components.
As the carbon source, it is possible to use sugars such as glucose, galactose, fructose, or starch hydrolysate; alcohols such as glycerol or sorbitol; or organic acids such as fumaric acid, citric acid or succinic acid.
As the nitrogen source, it is possible to use inorganic ammonium salts such as ammonium sulfate, ammonium chloride or ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; or aqueous ammonia.
It is desirable to allow required substances such as vitamin B1 and L-isoleucine or yeast extract to be contained in appropriate amounts as organic trace nutrients. Other than the above, potassium phosphate, magnesium sulfate, iron ion, manganese ion and the like are added in small amounts, if necessary.
Cultivation is preferably carried out under an aerobic condition for 16-72 hours. The cultivation temperature is controlled at 25C. to 45C., and pH is controlled at 5-8 during cultivation. Inorganic or organic, acidic or alkaline substances as well as ammonia gas or the like can be used for pH adjustment.
Collection of shikimic acid from a fermented liquor is usually carried out by combining an ion exchange resin method, a precipitation method and the like.